Antibodies to natural killer stimulatory factor

ABSTRACT

This application relates to antibodies reactive with a novel homogenous human cytokine, Natural Killer Stimulator Factor (NKSF), having the ability to induce the production of gamia interferon in vitro in human peripheral blood lymphocytes, and a pharmaceutical preparation containing such antibodies.

This is a division of application Ser. No. 09/325,958, filed Jun. 4,1999; which was a division of application Ser. No. 08/858,000, filed May16,1997, now abandoned; which was a continuation of application Ser. No.08/403,013, filed Mar. 13, 1995 and issued as U.S. Patent No. 5,648,467;which was a division of application Serial No. 07/584,941 filed Sep. 18,1990 and issued as U.S. Pat. No. 5,457,038, which was acontinuation-in-part of Ser. No. 07/307,817, filed Feb. 7, 1989, nowabandoned; which was a continuation-in-part of Ser. No. 07/269,945,filed Nov. 10, 1988, now abandoned, all of which are incorporated hereinby reference.

The present invention relates to a novel cytokine that stimulates thefunction of natural killer cells and other cells of the immune system,and to processes for obtaining the factor in homogeneous form andproducing it by recombinant genetic engineering techniques.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are a subset of lymphocytes active in theimmune system and representing an average 15% of mononuclear cells inhuman peripheral blood [G. Trinchieri and B. Perussia, Lab. Invest., 50:489 (1984)]. Among the surface markers used to identify human NK cellsis a receptor binding with low affinity to the Fc fragment of IgGantibodies, such as Fc-gamma receptor III or CD16 antigen [B. Perussiaet al, J. Immunol., 133:180 (1984)]. NK cells have been demonstrated toplay an important role in vivo in the defense against tumors, tumormetastases, virus infection, and to regulate normal and malignanthematopoiesis.

A growing family of regulatory proteins that deliver signals betweencells of the immune system has been identified. These regulatorymolecules are known as cytokines. Many of the cytokines have been foundto control the growth, development and biological activities of cells ofthe hematopoietic and immune systems. These regulatory molecules includeall of the colony-stimulating factors (GM-CSr, G-CSF, M-CSF, and multiCSF or interleukin-3), the interleukins (I-1 through IL-11), theinterferons (alpha, beta and gamma), the tumor necrosis factors (alphaand beta) and leukemia inhibitory factor (LIF). These cytokines exhibita wide range of biologic activities with target cells from bone marrow,peripheral blood, fetal liver, and other lymphoid or hematopoieticorgans. See, e.g., G. Wong and S. Clark, Immunology Today, 2(5):137(1988).

The biochemical and biological identification and characterization ofcertain cytokines was hampered by the small quantities of the naturallyoccurring factors available from natural sources, e.g., blood and urine.Many of the cytokines have recently been molecularly cloned,heterologously expressed and purified to homogeneity. [D. Metcalf, “TheMolecular Biology and Functions of the Granulocyte-Macrophage ColonyStimulating Factors,” Blood, 67(2):257-267 (1986).] Among thesecytokines are gamma interferon, human and murine GM-CSF, human G-CSF,human CSF-1 and human and murine IL-3. Several of these purified factorshave been found to demonstrate regulatory effects on the hematopoieticand immune systems in vivo, including GM-CSF, G-CSF, IL-3 and IL-2.

There remains a need in the art for additional proteins purified fromtheir natural sources or otherwise produced in homogeneous form, whichare capable of stimulating or enhancing immune responsiveness and aresuitable for pharmaceutical use.

BRIEF SUMMARY OF THE INVENTION

In one aspect the present invention provides a novel human naturalkiller stimulatory factor called NKSF, which is substantially free fromother mammalian proteins. Active NKSF has an apparent molecular weightof approximately 70-80 kD. Pure preparations of NKSF reveal the presenceof two polypeptides, subunits of approximately 40 kD and 30 kD, which,when associated, yield active NKSF. It is presently speculated that NKSFis a heterodimer formed by association of both the larger and smallersubunits through one or more disulfide bonds. This apparentheterodimeric structure can be generated by association of the twoindividual subunits.

The active, approximately 70-80 kD, NKSF is further characterized bycontaining all or a portion of the amino acid sequences of FIG. 1 (SEQID NO:4) and/or FIG. 2 (SEQ ID NO:6). Additionally, one or more of ninesequences of amino acids is present in the primary sequence of eitherthe larger or smaller of the NKSF subunits. These nine amino acidfragments are listed and discussed in detail below (SEQ ID NOS:7-15).

The larger subunit polypeptide of NKSF is characterized by having anapparent molecular weight of 40 kD. This subunit is furthercharacterized by having the same or substantially the same amino acidsequence as described in FIG. 1 (SEQ ID NO:4), containing the N-terminalsequence:

Ile-Trp-Glu-Leu-Lys-Lys-Asp-Val-Tyr-Val-Val-Glu-Leu-Asp-Trp-Tyr-Pro-Asp-Ala-Pro-Gly-Glu-Met(SEQ ID NO: 1). This N-terminal amino acid sequence corresponds to aminoacids #23-45 of FIG. 1. This polypeptide is further characterized bycontaining six of the nine amino acid fragments.

The smaller polypeptide subunit of NKSF is characterized bv an apparentmolecular weight of approximately 30-35 kD. Two cDNA sequences have beenidentified for the smaller subunit. The shorter of the two sequences issubstantially contained within the longer sequence in plasmidp35nksf14-1-1, illustrated in FIG. 2. The smaller subunit is furthercharacterized by having the same or substantially the same amino acidsequence as described in FIG. 2 (SEQ ID NO:6), containing the followingN-terminal sequence:

Arg-Asn-Leu-Pro-Val-Ala-Thr-Pro-Asp-Pro-Gly-Met-Phe-Pro (SEQ ID NO:2).This fragment corresponds to underlined amino acids #57-70 of thep35nksf14-1-1 clone.

This smaller polypeptide is further characterized by containing three ofthe nine fragments of amino acids identified by underlining in FIG. 2(SEQ ID NOS: 10-12).

NKSF displays biological activity in inducing the production of gammainterferon in vitro in human peripheral blood lymphocytes (PBLs). Inhomogeneous form, NKSF is characterized by a specific activity ofgreater than 1×10⁷ dilution units per milligram in the gamma interferoninduction assay, described in detail below.

In addition to the induction of gamma interferon in PBLs, NKSFdemonstrates the following biological activities:

(1) biological activity in a granulocyte-macrophage colony stimulatingfactor (GM-CSF) inducing assay with PBLs;

(2) biological activity in activating Natural Killer (NK) cells to killleukemia and tumor-derived cells;

(3) biological activity in a tumor necrosis factor (TNF) inducing assaywith phytohemagglutinin (PHA)-activated T lymphocytes;

(4) co-mitogenic activity with peripheral blood T lymphocytes; and

(5) synergizes with IL-2 in inducing γ IFN production in PBLs andmaintaining PBL proliferation.

Another aspect of the invention includes DNA sequences comprising cDNAsequences encoding the expression of a human NKSF polypeptide, a humanNKSF larger subunit polypeptide, and a human NKSF smaller subunitpolypeptide. Such sequences include a sequence of nucleotides encodingone or more of the subunits and peptide sequences described above.

Also provided by the present invention is a vector containing a DNAsequence encoding NKSF or a subunit of NKSF in operative associationwith an expression control sequence. Host cells transformed with suchvectors for use in producing recombinant NKSF or its recombinantsubunits are also provided by the present invention.

As still a further aspect of the present invention, there is providedrecombinant NKSF protein. This protein is free from other mammalianproteinaccous materials and is characterized by the presence of a DNAsequence encoding one or more of the above-described subunits or peptidefragments containing one or more of the above-described physical,biochemical or biological activities or characteristics.

Another aspect of this invention provides pharmaceutical compositionscontaining a therapeutically effective amount of homogeneous orrecombinant NKSF, or an effective amount of one or both of the subunitsof NKSF, or of one or more of the peptide fragments thereof. Thesepharmaceutical compositions may be employed in methods for treatingcancer, viral infections, such as AIDS, bacterial infections, and otherdisease states responsive to the enhanced presence of gamma interferonor GM-CSF production. Thus, generally this factor may be employed in thetreatment of diseases in which stimulation of immune function might bebeneficial.

A further aspect of the invention, therefore, is a method for treatingcancer and/or other pathological states which may benefit from enhancednatural killer cell functions by administering to a patient atherapeutically effective amount of NKSF or one or both of its subunitsor peptide fragments thereof in a suitable pharmaceutical carrier. Thesetherapeutic methods may include administering simultaneously orsequentially with NKSF or one or more of its subunits or peptidefragments an effective amount of at least one other cytokine,hematopoietin, interleukin, growth factor, or antibody. Specifically,the administration of NKSF or one or more of its subunits with IL-2 hasdemonstrated synergistic effects. Because of the synergy with IL-2 invitro, this interleukin might be particularly effective in combinationwith NKSF.

Still a further aspect of the present invention is a process forproducing homogeneous NKSF, or a subunit thereof from a human cell lineproducing NYSF or a subunit thereof in admixture with other proteins andpolypeptides. This process of production provided by the presentinvention includes culturing selected cells capable of producing NKSF,its subunits, or peptide fragments thereof to obtain conditioned mediumand purifying the conditioned medium through five primary purificationsteps.

The vectors and transformed cells of the invention are employed inanother aspect, a novel process for producing recombinant human NKSFprotein, a subunit thereof or peptide fragments thereof. In this processa cell line transformed with a DNA sequence encoding on expression NKSFprotein, a subunit thereof or a peptide fragment thereof in operativeassociation with an expression control sequence therefore is cultured.This claimed process may employ a number of known cells as host cellsfor expression of the polypeptide. Presently preferred cell lines aremammalian cell lines and bacterial cells.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description of preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4)sequence of the 40 kD subunit of NKSF.

FIG. 2 is a nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO:6)sequence of the 30 kD subunit of NKSF.

DETAILED DESCRIPTION OF THE INVENTION

The novel human natural killer cell stimulatory factor, NKSF, providedby the present invention is a homogeneous protein or proteinaceouscomposition substantially free of association with other mammalianproteinaceous materials.

Natural killer stimulatory factor has an apparent molecular weight ofapproximately 70-80 kD as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under non-reducingconditions. This 70-80 kD peptide is active in a gamma interferoninduction assay.

Under reducing conditions in SDS-PAGE, the 70-80 kD band yields twosmaller subunits with apparent molecular weights of approximately 40 kD(larger subunit) and approximately 30-35 kD (smaller subunit). For bothsubunits individually, the biological activity in the same gammainterferon induction assay is substantially lost compared to that of thenative 70-80 kD species. The amino terminal sequences identified abovewere originally detemined from the 40 kD reduced species and the 30-35kD reduced species believed to be the subunits of the NKSF heterodimer.It is presently believed that NKSF is a disulfide-honded heterodimer ofthe larger and smaller subunits. However, it is also possible that oneor both of these subunits, when present alone, may have biologicalactivity.

NKSF is, at least in part, an anionic glycoprotein. Under isoelectricfocusing, two species of the NKSF are observed having isoelectric pointsof 4.3 and 4.8. It is presently speculated that the two species differin glycosylation patterns.

NKSF is primarily characterized by biological activity in the gammainterferon induction assay described in detail in Example 8 below. Amongits other biological activities include the ability to induce GM-CSFproduction by human peripheral blood lymphocytes. [See, e.g., publishedPCT application WO86/00639 for additional information on GM-CSF]. NKSFalso has an enhancing effect on the mitogenic activity of variousmitogens, such as lectins and phorbol diesters, on peripheral blood Tlymphocytes and has a growth promoting effect on activated humantonsillar B cells. NKSF has also been observed to enhance NK cellfunctions to kill leukemia and tumor-derived cells in vitro. using aspontaneous cell cytotoxicity assay and an antibody dependent cellcytotoxicity (ADCC) assay.

In a spontaneous cell cytotoxicity assay, human peripheral bloodlymphocytes or purified NK cells are incubated in the presence of NKSFfor a period of 8 to 18 hours. Lymphocytes and NK cells are then assayedin a standard ⁵¹Cr-release assay for their ability to lyse target cellssuch as leukemia cell lines, tumor-derived cell lines, or virus-infectedfibroblasts. NKSF dramatically increases the ability of NK cells to lysesuch target cells at a level comparable to that obtained with interferonalpha and IL-2, well known activators of NK cell cytotoxic activity[See, e.g., G. Trinchieri et al, J. Exp. Med., 147:1314 (1978) and G.Trinchieri et al, J. Exp. Med., 160:1146 (1984)].

In an ADCC assay target cancer cells are coated with antibodies capableof binding to the Fc receptor on NK cells, e.g., igG₂a, IgG₃ and thelike. In preliminary assays, the presence of NKSF appears to enhance thekilling activity of the NK cells for the coated tumor cells in ADCC.[See, e.g., L. M. Weiner et al, Cancer Res., 48:2568-2573 (1988); P.Hersey et al, Cancer Res., 46:6083-6090 (1988); and C. J. Hansik et al,Proc. Natl. Acad. Sci., 83:7893-97 (1986) for additional information onADCC.]

Preliminary analysis of NKSF in a B-cell growth factor assay usingnormal human B cells stimulated with goat anti-human IgM antibody(anti-μ) coupled to beads indicates that NKSF may also be characterizedby B cell growth factor activity. In this assay the antibody directedagainst the IgM immunoglobulin on the surface of the B cell activatesthe B cell and causes it to become responsive to B cell growth factors.[See, C-T K. Tseng et al, J. Immunol., 140:2305-2311 (1988)]. Suchantibodies are commercially available.

NKSF was originally detected in the conditioned medium of the human cellline, RPMI 8866, a commercially available cell line [University ofPennsylvania Cell Center] which produces a mixture of lymphokines. Thisfactor may also be produced by other Epstein Barr virus-transformedlymphoblastoid cell lines or from other human cell lines. The RPMI 8866cell line produces the factor spontaneously, but the level of productioncan be enhanced by treating the cell line with phorbol esters, such asphorbol dibutyrate. The cells deprived of serum for 48 hours stillproduce NKSF along with other lymphokines. Procedures for culturing RPMI8866 (see Example 1) or another cell source of NKSF are known to thoseof skill in the art.

The purification technique employed in obtaining NKSF from cells whichnaturally produce it, uses the following steps. These steps includepurification through an ion exchange column, e.g., QAE Zeta preparativecarnidge [LKB Pharmacea], which indicates that the NKSF protein isanionic. The second purification step is a lentil lectin column whichdemonstrates that NKSF is, at least in part, a glycoprotein. The eluatefrom the lentil lectin column is further purified through ahydroxylapatite column, followed by a heparin sepharose column and afast protein liquid chromatography (FPLC) Mono-Q column. The NKSF fromRPMI 8866 eluted as a single peak in each of the three latter columns. Aremaining protein contaminant of about 37 kD is removed by gelfiltration chromatography alone or reverse phase HPLC and gel filtrationchromatography. The resulting purified homogeneous NKSF was assayed forbiological activity in the gamma interferon induction assay of Example 8and demonstrated a specific activity of greater than 1×10⁷ dilutionunits per milligram.

Thus, the homogeneous NKSF may be obtained by applying the abovepurification procedures, which are described in detail in Example 2 tothe conditioned medium of RPMI 8866 or other sources of human NKSF.

NKSF, one or both of its subunits, or peptide fragments thereof may alsobe produced via recombinant techniques, e.g., by culturing undersuitable conditions a host cell transfected with DNA sequences encodingthe larger and/or smaller subunit in operative association with aregulatory control sequence capable of directing expression thereof.

The DNA sequences for cloned NKSF and its subunits were originallyisolated by preparing tryptic digests of the homogeneous polypeptide.For example, the nine tryptic fragments originally found in NKSF areidentified below: Fragment 1: Leu-Thr-Ile-Gln-Val (SEQ ID NO:7) Fragment2: Lys-Tyr-Glu-Asn-Tyr-Thr (SEQ ID NO:8) Fragment 3: Ile-Trp-Glu-Leu-Lys(SEQ ID NO:9) Fragment 4: Leu-Met-Asp-Pro-Lys (SEQ ID NO:10) Fragment 5:Val-Met-Ser-Tyr-Leu-Asn-Ala (SEQ ID NO:11) Fragment 6:Ala-Val-Ser-Asn-Met-Leu-Gln-Lys (SEQ ID NO:12) Fragment 7:Asn-Ala-Ser-Ile-Ser-Val (SEQ ID NO:13) Fragment 8: Thr-Phe-Leu-Arg (SEQID NO:14) Fragment 9: Asp-Ile-Ile-Lys-Pro-Asp-Pro-Pro-Lys (SEQ ID NO:15)

Fragments 4 (SEQ ID NO:10), 5 (SEQ ID NO:11) and 6 (SEQ ID NO:12) havebeen identified as being located within the smaller or 30 kD subunit.These sequences correspond to the underlined amino acids #179-184,246-252, and 81-86, respectively, of the p35nksf14-1-1 clone illustratedin FIG. 2. Fragments 1-3 (SEQ ID NOS:7-9) and 7-9 (SEQ ID NOS:13-15)have been identified as being located within the larger, 40 kD, NKSFsubunit. Amino acid sequences corresponding to Fragment 1 (amino acids#75-79); Fragment 2 (amino acids #219-224); Fragment 3 (amino acids#23-27); Fragment 7 (amino acids #303-308); Fragment 8 (amino acids#127-130); and Fragment 9 (amino acids #231-239) are underlined in FIG.1 (SEQ ID NO:4). Additionally, the amino terminal sequences of thelarger and smaller subunits of NKSF were identified as described belowin Example 5 and are underlined in FIG. 1 (#23-45 of SEQ ID NO:4) andFIG. 2 (#57-70 of SEQ ID NO:6), respectively.

Oligonucleotide probes were synthesized using the genetic code topredict all possible sequences that encode the amino acid sequences ofthese tryptic digestion products of NKSF. The same procedure may befollowed by constructing probes from the above-identified amino terminalsequences of the two subunits of NKSF. The NKSF subunit genes can beidentified by using these probes to screen a human genomic library.Alternatively, the mRNA from RPMI-8866 or another cell source of NKSFcan be used to make a cDNA library which can be screened with the probesto identify the cDNAs encoding the polypeptides of the NKSF large andsmall subunits. Once the cDNAs were identified, they were introducedinto an expression vector to make an expression system for NKSF, or oneor both of its subunits.

By such use of recombinant techniques, DNA sequences encoding thepolypeptides of the NKSF large and small subunit were obtained, whichcontain DNA sequences encoding the tryptic fragments or the aminoterminal sequences identified above.

One NKSF clone, named pNK40-4, has the DNA (SEQ ID NO:3) and amino acid(SEQ ID NO:4) sequences presented in FIG. 1 below and codes for all or aportion of the larger NKSF subunit:

This cloned sequence in plasmid pNK40-4 was deposited with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. on Jul.31, 1990 under accession number 40854. A prior partial clone of thislarger fragment, containing the N-terminal non-coding region throughsequence containing up to approximately nucleotide #888 was called pNK-6and deposited with the ATCC on Feb. 3, 1989 under ATCC No.40545. Anotherpartial clone of the larger subunit, pNK162 was sequenced and containsthe sequence from nucleotides #643 to 2362 of SEQ ID NO:3 in FIG. 1.This clone is maintained at the labs of Genetics Institute, Inc.

Two independent cDNA clones were identified which encode the sequence ofthe small (30-35 kD) subunit of NK.SF. The longer clone (designatedp35nksf14-1-1) is shown in FIG. 2 (SEQ ID NO:5). The shorter clone(designated p35nksf9-1-1) begins at nucleotide #133 (indicated by *) andends at nucleotide #1335 (indicated by *) of FIG. 2 and the depositedsequence. Between those two nucleotides, the smaller clone is identicalto the sequence of FIG. 2 except for 5 nucleotide changes in the 3′non-coding region. This shorter clone thus has a coding sequencebeginning with Met (amino acid #35) in FIG. 2. The additional sequenceat the 5′ end of p35nksf14-1-1 encodes an in-frame initiation codon(ATG) 34 residues 5′ of the operative initiation codon in p35nksf9-1-1.

Both of these clones encode all of the peptide sequences identified inthe tryptic digest of purified NKSF, which were not found in the 40 kDsubunit protein, as well as the amino terminal sequence of the purified30 kD subunit. These sequences are underlined in SEQ ID NO:6 of FIG. 2.The clones contain the coding sequence for two possible versions of the30-35 kD subunit of NKSF depending on whether translation begins withMet #1 or Met #35 in FIG. 2. However, because the 30-35 kD proteinsubunit of NKSF is believed to be generated by cleavage following Ala(amino acid #56), both sequences should yield the same mature protein.The sequence of p35nksf14-1-1 was deposited with the ATCC on Sep. 11,1990 under accession number 40886.

Sequence from p35nksf9-1-1 (from the Pst I site underlined in FIG. 2 tothe Pst I site in the Bluescript polylinker sequence), when introducedinto Cos cells in the expression vector pEMC3(l) along with a plasmidexpressing the 40 kD subunit yielded biologically active NKSF. Thismaterial was active in the same bioassays used to test natural NKSF asdiscussed below. This sequence may be obtained from p35nksf14-1-1 bydigestion with Pst I. Alternatively the cloned sequence of plasmidp35nksf9-1-1, containing the shorter 30-35 kD subunit sequence, is beingmaintained at the laboratories of Genetics Institute, CambridgePark,Mass. and will be made available to the public upon grant of the patent.

A cDNA suitable for expression of the longer version of the 30-35 kDsubunit may be obtained from the p35nksf14-1-1 deposited clone bydigestion with SalT and NotI. The longer 30-35 kD subunit contains anearlier Met (amino acid #1 in SEQ ID NO:6) codon, additional 5′ codingand non-coding sequences as well as 3′ non-coding sequence. The sequencefrom Met (amino acid #35) to the N-terminus of the mature protein(encoded by both cDNAs) encodes a sequence which resembles a signalpeptide and may direct the proper folding and/or secretion of thesubunit. It is therefore possible that the longer 30-35 kD subunitsequence may be more efficiently expressed and secreted by the Cos cellsthan the shorter version. It may also fold differently, therebyconferring NKSF activity independent of the presence of the 40 kDsubunit.

FIG. 2 indicates the placement of polylinker sequence in the depositedclone, as well as the first and last nucleotide of the larger andsmaller versions of this subunit. Also indicated in the sequence are the5′ PstI site for obtaining the sequence of the small subunit which hasbeen expressed and underlined tryptic fragment sequences.

Allelic variations of DNA sequences encoding the peptide sequences andthe large and small subunits described above are also included in thepresent invention as well as analogs or derivatives thereof.

Thus the present invention also encompasses these novel DNA sequences,free of association with DNA sequences encoding other primate proteins,and coding on expression for NKSF polypeptides, including those of itslarge and small subunits. These DNA sequences include those containingone or more of the above-identified DNA and peptide sequences and thosesequences which hybridize under stringent hybridization conditions [see,T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold SpringHarbor Laboratory (1982), pages 387 to 389] to the DNA sequences. Anexample of one such stringent hybridization condition is hybridizationat 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C. for anhour. Alternatively an exemplary stringent hybridization condition is in50% formamide, 4×SSC at 42° C.

DNA sequences which hybridize to the sequences for NKSF or its subunitsunder relaxed hybridization conditions and which code on expression forNKSF peptides having NKSF biological properties also encode novel NKSFpolypeptides. Examples of such non-stringent hybridization conditionsare 4×SSC at 50° C. or hybridization with 30-40% formamide at 42° C. Forexample, a DNA sequence which shares regions of significant homology,e.g., sites of glycosylation or disulfide linkages, with the sequencesof NKSF and encodes a protein having one or more NKSF biologicalproperties clearly encodes a NKSF polypeptide even if such a DNAsequence would not stringently hybridize to the NKSF sequences.

Similarly, DNA sequences which code for NKSF polypeptides coded for bythe sequence of NKSF, but which differ in codon sequence due to thedegeneracies of the genetic code or allelic variations(naturally-occurring base changes in the species population which may ormay not result in an amino acid change) are also encompassed by thisinvention. Variations in the DNA sequence of NKSF which are caused bypoint mutations or by induced modifications to enhance the activity,half-life or production of the polypeptides encoded thereby are alsoencompassed in the invention.

NKSF polypeptides may also be produced by known conventional chemicalsynthesis. Methods for constructing the polypeptides of the presentinvention by synthetic means are known to those of skill in the art. Thesynthetically-constructed NKSF polypeptide sequences, by virtue ofsharing primary, secondary, or tertiary structural and conformationalcharacteristics with NKSF polypeptides may possess NKSFbiological-properties in common therewith. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedNKSF polypeptides in therapeutic and immunological processes.

The NKSF polypeptides provided herein also include factors encoded bysequences similar to those of purified homogeneous and recombinant NKSFprotein, or the subunit polypeptides, but into which modifications arenaturally provided or deliberately engineered. Modifications in thepeptides or DNA sequences can be made by one skilled in the art usingknown techniques. Modifications of interest in the NKSF sequences mayinclude the replacement, insertion or deletion of a selected amino acidresidue in the coding sequences. Mutagenic techniques for suchreplacement, insertion or deletion are well known to one skilled in theart. [See, e.g., U.S. Pat. No. 4,518,584.]

Other specific mutations of the sequences of the NKSF polypeptide or thesubunit polypeptides described herein may involve modifications of aglycosylation site. The absence of glycosylation or only partialglycosylation results from amino acid substitution or deletion at anyasparagine-linked glycosylation recognition site or at any site of themolecule that is modified by addition of O-linked carbohydrate. Anasparagine-linked glycosylation recognition site comprises a tripeptidesequence which is specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are eitherasparagine-X-threonine or asparagine-X-serine, where X is usually anyamino acid. A variety of amino acid substitutions or deletions at one orboth of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.

Expression of such altered nucleotide sequences produces variants whichare not glycosylated at that site.

Other analogs and derivatives of the sequence of NKSF or of its subunitswhich would be expected to retain NKSF activity in whole or in part mayalso be easily made by one of skill in the art given the disclosuresherein. One such modification may be the attachment of polyethyleneglycol onto existing lysine residues or the insertion of a lysineresidue into the sequence by conventional techniques to enable theattachment. Such modifications are believed to be encompassed by thisinvention.

The present invention also provides a method for producing NKSFpolypeptides. The method of the present invention involves culturing asuitable cell or cell line, which has been transformed with a DNAsequence coding on expression for an NKSF polypeptide or subunit, underthe control of known regulatory sequences. Preferably DNA sequences forboth subunits are transformed into a host cell.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See, e.g., Gething and Sambrook, Nature. 293:620-625(1981), or alternatively, Kaufman et al, Mol. Cell. Biol.,5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446.Expression of two different DNAs simultaneously in CHO cells has beendescribed, for example, in published PCT International ApplicationWO88/08035. Other suitable mammalian cell lines, are the monkey COS-1cell line, and the CV-1 cell line, originally developed at the WistarInstitute, Philadelphia, Pa.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, MC1061 and strains used in the following examples) are well-knownas host cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas, other bacilli and the like may also be employedin this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

The present invention also provides vectors for use in the method ofexpression of novel NKSF polypeptides. These vectors contain the novelNKSF DNA sequences which code for NKSF polypeptides of the invention,including the subunit polypeptides. Alternatively, vectors incorporatingmodified sequences as described above are also embodiments of thepresent invention and useful in the production of NKSF polypeptides. Thevector employed in the method also contains selected regulatorysequences in operative association with the DNA coding sequences of theinvention and capable of directing the replication and expressionthereof in selected host cells.

Thus NKSF, purified to homogeneity from cell sources or producedrecombinantly or synthetically, may be used in a pharmaceuticalpreparation or formulation to treat cancer or other disease states whichrespond to enhanced NK cell activity or increased in vivo production ofgamma interferon or GM-CSF. Such pathological states may result fromdisease, exposure to radiation or drugs, and include for example,leukopenia, bacterial and viral infections, anemia, B cell or T celldeficiencies including immune cell or hematopoietic cell deficiencyfollowing a bone marrow transplantation. Therapeutic treatment of cancerand other diseases with these NKSF polypeptide compositions may avoidundesirable side effects caused by treatment with presently availabledrugs. The NKSF polypeptide compositions according to the presentinvention may also be used in the treatment of Acquired ImmunodeficiencySyndrome (AIDS) and other viral infections, particularly non-responsiveviral infections, as well as bacterial infections.

It may also be possible to employ one or both of the subunitpolypeptides of NKSF or peptide fragments thereof in such pharmaceuticalformulations.

The polypeptides of the present invention may also be employed, alone orin combination with other cytokines, hematopoietins, interleukins,growth factors or antibodies in the treatment of cancer or other diseasestates. For example, NKSF polypeptides have been shown to have asynergistic effect when administered in connection with IL-2. This isexpected to be useful in the treatment of infections, particularly viralinfections and cancers. Other uses for these novel polypeptides are inthe development of monoclonal and polyclonal antibodies generated bystandard methods for diagnostic or therapeutic use.

Therefore, as yet another aspect of the invention are methods andtherapeutic compositions for treating the conditions referred to above.Such compositions comprise a therapeutically effective amount of theNKSF protein or a subunit polypeptide or therapeutically effectivefragment thereof of the present invention in admixture with apharmaceutically acceptable carrier. This composition can besystemically administered parenterally. Alternatively, the compositionmay be administered intravenously. If desirable, the composition may beadministered subcutaneously. When systematically administered, thetherapeutic composition for use in this invention is in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such a pharmaceutically acceptable protein solution, having dueregard to pH, isotonicity, stability and the like, is within the skillof the art.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician consideringvarious factors which modify the action of drugs, e.g. the condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. Generally, the dailyregimen should be in the range of 1-1000 micrograms of NKSF protein orsubunit thereof or 50 to 5000 units (i.e., one unit per ml being theconcentration of protein which leads to half maximal stimulation in thegamma interferon induction assay) of protein per kilogram of bodyweight.

The therapeutic method and compositions of the present invention mayalso include co-administration with other human factors. Exemplarycytokines or hematopoietins for such use include the known factors IL-1,IL-2 and IL-6 particularly. [See, e.g., PCT publications WO85/05124, andWO88/00206; and European patent application 0,188,864.]. Other potentialcandidates for participation in NKSF therapy may also include IL-4,G-CSF, CSF-1, GM-CSF, IL-3, IL-11 or erythropoietin. Growth factors likeB cell growth factor, B cell differentiation factor, or eosinophildifferentiation factors may also prove useful in co-administration withNKSF.

Similarly, administration of NKSF or a subunit or fragment thereof withor prior to administration of an antibody capable of binding to the Fcreceptor on NK cells may enhance ADCC therapy directed against tumors.The dosage recited above would be adjusted to compensate for suchadditional components in the therapeutic composition. Progress of thetreated patient can be monitored by conventional methods.

The following examples illustratively describe the purification andcharacteristics of homogeneous human NKSF and other methods and productsof the present invention. These examples are for illustration and do notlimit the scope of the present invention.

EXAMPLE 1 PREPARATION OF SERUM-FREE RPMI 8866 CELL-CONDITIONED MEDIUM

The human B-lymphoblastoid cell line RPMI 8866 was maintained in RPMI1640 medium containing 5% heat-inactivated fetal calf serum (FCS). Forpreparation of serum free conditioned medium, cells were washed andsuspended (10⁶ cells/ml) in serum free RPMI 1640 medium containing 10⁻⁷M phorbol-12-13-dibutvrate (PdBU) and cultured for 48 hours at 37° C.,5% CO₂. The cell free supematants were harvested by filtration through a0.2 μm filter [Durapore® hydrophilic cartridge filter, Millipore,Bedford, Mass. and Tween-20 and phenylmethylsulfonyl-fluoride (PMSF)were added to 0.02% and 0.1 mM, respectively. The cell conditionedmedium was then concentrated 50 fold under pressure using anultra-filtration cartridge [Spiral-Wound, S1, Amicon, Danvers, Mass.].

EXAMPLE 2 PURIFICATION OF NKSF FROM CONDITIONED MEDIUM

The following procedures are presently employed to obtain homogeneousNKSF protein from RPMI 8866 conditioned medium, as described in Example1 above.

a. Anion Exchange Cartridge Chromatography

Two liters of the crude concentrated conditioned medium was diluted withdistilled water to a conductivity of 6m Os/cm and adjusted to pH 8 with1 M Tris-HCl buffer (pH 8). The concentrate was then applied to five QAEZetaprep 250 cartridges [Pharmacia] connected in parallel and previouslyequilibrated with 0.1 M Tris-HCl buffer (pH 8) at a flow rate of 150ml/min. Unless otherwise cited, all the buffers used for purificationcontained 0.02% Tween-20 and 0.1 mM PMSF. The cartridges were washedwith 3 liters of 0.1 M Tris-HCI buffer (pH 6.8) followed by washing with1.5 liters of 0.5 M NaCl in 0.1 M Tris-HCl buffer (pH 6.8) and 300 mlfractions were collected. The NKSF activity was eluted with the 0.5 MNaCl-containing wash.

b. Lentil-Lectin Sepharose Chromatography

Pooled NKSF-containing fractions from two separate QAE Zetaprep elutionswere pooled and applied directly to a column (2.5×15 cm) oflentil-lectin Sepharose 4B [Pharmacia] which has been equilibrated with20 mM Tris-HCl buffer (pH 7.2). After washing with five column volumesof equilibration buffer, the column was eluted with three column volumesof 20 mM Tris-HCI buffer (pH 7.2) containing 0.2 Mα-methyl-D-mannopyranoside [Sigma] and 0.5 M NaCl. Approximately half ofthe NKSF activity was bound by the column and was recovered in thefractions eluted with α-methyl-D-mannopyranoside.

c. Hydroxylapatite Chromatography

Concentrated material from the pool of NKSF activity which bound to thelentil-lectin Sepharose column was dialyzed against 1 mM potassiumphosphate buffer (pH 6.8) containing 0.1 mM CaCl₂ and 0.15 M NaCl andapplied to a Biogel HT [BioRad] column (2×5 cm) previously equilibratedwith 1 mM potassium phosphate buffer (pH 6.8) containing 0.1 mM CaCl₂.The column was washed with five column volumes of equilibration bufferand eluted with 100 ml of a linear gradient from 1 mM to 400 mMpotassium phosphate buffer (pH 6.8) containing 0.15 M NaCl. 4 mlfractions were collected and tested for NKSF activity. A single peak ofactivity emerged from the column between the approximately 200 mM and300 mM potassium phosphate.

d. Heparin-SeDharose Chromatooraphy

Eluted NKSF-containing fractions from the Biogel HT column were pooledand dialyzed against 20 mM sodium phosphate buffer (pH 7.2) and appliedto a Heparin Sepharose [Pierce, Rockford, Ill.] column (1×10 cm). Thecolumn was washed with five column volumes of 20 mM sodium phosphatebuffer (pH 7.2) and eluted with the same buffer containing 1 M NaCl. 3ml fractions were collected and NKSF activity measured. Essentially allof the activity was bound by the Heparin column and recovered in the 1 MNaCl wash.

e. Mono Q Chromatography

Pooled fractions from the Heparin Sepharose column were dialyzed against20 mM Tris-HCl buffer (pH 6.8) containing 1% ethylene glycol and 0.1 mMPMSF but no Tween-20 (buffer A) and concentrated to 2 ml using a stirredcell [Amicon] with a YM 10 membrane. The sample was applied to a Mono Q(5/5) column [Pharmacia-FPLC apparatus] and eluted with a lineargradient from 0 M to 1 M NaCl in buffer A (pH 6.8). 0.5 ml fractionswere collected and tested for NKSF activity. The activity emerged fromthe column as a single peak between approximately 220 mM and 270 mMNaCl.

f. Gel Filtration Chromatography

Pooled fractions containing NKSF activity from the Mono Q column wereconcentrated to 100 microliters by Speedvac Concentrator [Savant,Farmingdale, N.Y.] and applied to a FPLC Superose 12 column.Chromatography was run with 50 mM sodium phosphate buffer (pH 7.2)containing 0.15 M NaCl, 1% ethylene glycol and 0.1 mM PMSF. Flow ratewas 0.6 ml/minute and 0.5 ml fractions were collected. NKSF protein (70kD) was separated from the approximately 37 kD protein contaminant.

Alternatively, the pooled Mono-Q fractions may be subjected toreverse-phase HPLC (C8 column) prior to the step (f) described above, toseparate the protein contaminant from the active 70 kD protein.

EXAMPLE 3 SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS

SDS-PAGE was performed according to the method of Laemmli [Laemmli, U.K., Nature, 227:680-685 (1970)] on 10% acrylamide slab gels (0.75 mmthickness). After electrophoresis the gels were either stained by thesilver-nitrate method using a silver staining reagents [BioRad] or cutinto 2 mm slices and eluted in 0.5 m! RPMI medium for 4 hours at 24° C.and assayed for NKSF activity. Apparent molecular weight was determinedwith protein standards, phospholipase b (94 kD), bovine serum albumin(67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), soybean trypsininhibitor (20 kD) and lactalbumin (14.4 kD).

SDS-PAGE analysis (non-reducing conditions) of the Mono Q columnfractions (Example 2, step (e)) beginning with several fractions whicheluted before the NKSF activity, continuing right through the activefractions and ending with fractions which eluted after the peak of NKSFactivity, revealed that the presence of two proteins (70 kD and 37 kD)correlated with the presence of NKSF activity in the various Mono Qfractions. The active fractions were rerun on a second non-reducing geland the proteins were eluted from the regions corresponding to the 70 kDand 37 kD bands and tested for NKSF activity. The activity allcorrelated with the 70 kD species indicating that this protein is NKSF.

The 70 kD species was eluted from the gel, lodinated using chloramine T[Sigma, St. Louis, Mo.] and rerun on a second SDS gel after boiling fortwo minutes in the presence of the reducing agent, β-mercaptoethanol(10%). Under these conditions, the 70 kD species resolved into twodistinct subunits of molecular weights 40 kD and 30 kD, indicating thatthe native NKSF may be a disulfide-bonded heterodimer of these subunitpolypeptides. Alternatively, NKSF may be a dimer formed by multiples ofthe larger or smaller subunits. The reduction of the native 70 kD NKSFappeared to destroy all of its ability to induce peripheral bloodlymphocyte production of gamma interferon.

EXAMPLE 4 RECOVERY OF PROTEIN

Starting with 500 liters of RPMI 8866 cell-free conditioned medium, thefinal pooled active fractions from the Mono Q column containedapproximately 10 μg of protein, estimated from the intensities of silverstaining by control proteins analyzed in parallel on the same gel.Approximately 6 μg of this corresponded to the 70 kD NKSF protein. Theestimated specific activity of the 70 kD NKSF is 1×10⁷ u/mg. The overallrecovery of NKSF activity in the preparation was 2%.

EXAMPLE 5 NKSF PROTEIN COMPOSITION

Homogeneous NKSF is reduced as described in the SDS-PAGE example aboveand digested with trypsin. Alternatively, non-reduced NKSF may beobtained from a reverse-phase HPLC column and digested with trypsin.Nine tryptic fragments are isolated having the following amino acidsequences:. Fragment 1 Leu-Thr-Ile-Gln-Val SEQ ID NO:7 Fragment 2Lys-Tyr-Glu-Asn-Tyr-Thr SEQ ID NO:8 Fragment 3 Ile-Trp-Glu-Leu-Lys SEQID NO:9 Fragment 4 Leu-Met-Asp-Pro-Lys SEQ ID NO:10 Fragment 5Val-Met-Ser-Tyr-Leu-Asn-Ala SEQ ID NO:11 Fragment 6Ala-Val-Ser-Asn-Met-Leu-Gln-Lys SEQ ID NO:12 Fragment 7Asn-Ala-Ser-Ile-Ser-Val SEQ ID NO:13 Fragment 8 Thr-Phe-Leu-Arg SEQ IDNO:14 Fragment 9 Asp-Ile-Ile-Lys-Pro-Asp-Pro-Pro-Lys. SEQ ID NO:15

Additionally, the amino acid sequences of the amino termini of eachsubunit of NKSF were determined from the isolated 40 kD and 30 kDspecies of NKSF after reduction, as described in Example 3. The aminoterminal sequence from the 40kD subunit was as follows:Ile-Trp-Glu-Leu-Lys-Lys-Asp-Val-Tyr-Val-Val-Glu-Leu-Asp-Trp-Tyr-Pro-Asp-Ala-Pro-Gly-Glu-Met(SEQ ID NO: 1). The amino terminal sequence above as well as Fragments1-3 (SEQ ID NOS:7-9) and 7-9 (SEQ ID NOS: 13-15) proved to be derivedfrom the amino acid sequence of the clone of larger subunit identifiedin FIG. 1.

The amino terminal sequence from the 30kD smaller subunit was asfollows: Arg-Asn-Leu-Pro-Val-Ala-Thr-Pro-Asp-Pro-Gly-Met-Phe-Pro (SEQ IDNO:2). Fragments 4 (SEQ ID NO: 10), 5 (SEQ ID NO:11) and 6 (SEQ ID NO:12) proved to be derived from the amino acid sequence of the clone ofthe smaller subunit identified in FIG. 2.

Probes consisting of pools of oligonucleotides or uniqueoligonucleotides are designed according to the method of R. Lathe, J.Mol. Biol., 183(1):1-12 (1985). The oligonuclectide probes aresynthesized on an automated DNA synthesizer.

Because the genetic code is degenerate (more than one codon can code forthe same amino acid) a mixture of oligonucleotides must be synthesizedthat contains all possible nucleotide sequences encoding the amino acidsequence of the tryptic fragment. It may be possible in some cases toreduce the number of oligonucleotides in the probe mixture based oncodon usage because some codons are rarely used in eukaryotic genes, andbecause of the relative infrequency of the dinucleotide CpG ineukaryotic coding sequences [see J. J. Toole et al, Nature, 312:342-347(1984)]. The regions of the amino acid sequences used for probe designare chosen by avoiding highly degenerate codons where possible. Theoligonucleotides are synthesized on an automated DNA synthesizer and theprobes are then radioactively labelled with polynucleotide kinase and³²P-ATP.

A cDNA encoding the small subunit of NKSF was identified by screening acDNA library (prepared in lambda Zap; Stratagene cloning systems, LaJolla, Calif.) made from polyadenylated RNA from PdBu induced 8866 cells(Univ. of Pennsylvania Cell Center) using established techniques (seeToole et al cited above). The screening was caried out usingoligonucleotides with sequence predicted by those tryptic peptides notcontained within the previously cloned cDNA coding for the 40 kD proteinas probes. Recombinants from this library are plated and duplicatenitrocellulose replicas made of the plates. The oligonucleotides arekinased with ³²p gamma ATP and hybridized to the replicas.

In particular two pools of oligonucleotides were synthesized based onthe peptide Val-Met-Ser-Tyr-Leu-Asn-Ala (SEQ ID NO: 11). The sequencesin one pool of 17mers were derived from the peptide sequenceMet-Ser-Tyr-Leu-Asn-Ala (SEQ ID NO: 16) and those in the second fromVal-Met-Ser-Tyr-Leu-Asn (SEQ ID NO: 17). Clones which hybridized to thefirst pool of oligonucleotides were hybridized with the second pool.Hybridizations were performed at 48° C. in a buffer containing 3M TMAC.Filters were subsequently washed in 3M TMAC, 50 mM Tris pH 8 at 50° C.[See K. A. Jacobs et al, Nucl. Acids Res., 16:4637-4650 (1988).]Duplicate positives were plaque purified. Two clones were identifiedwhich hybridized to both pools, p35nksf9-1-1, and p35nksf14-1-1,described above.

The sequence and computer translations of cDNA clone p35nksf14-1-1 isshown in FIG. 2. It includes all the peptide sequences identified intryptic digests of purified NKSF not found in the 40 kD subunit protein(underlined) as well as the amino terminal sequence of the purified 30kD subunit (underlined).

To obtain a full length cDNA clone for the 40 kD subunit of NKSF, cDNAthat had been previously prepared from 8866 polyadenylated RNA wascloned into λZAP as described above. Two hundred thousand recombinantsfrom this library were plated, duplicate nitrocellulose filters wereprepared and screened with a random primed ³²p labeled DNA fragment, thesequence of which is within pNK-6. The probing was done using standardstringent hybridization and washing conditions. Three duplicate positiveplaques resulted from this screen. The plaques were replated andreprobed using the above probe and conditions to clonally isolate theplaques. The three isolates were then probed with a ³²p end-labeledoligo dT probe (pd(T)₁₂₋₁₈, Pharmacia). This hybridization was done in6×SSC, 5× Denhardt's solution, and carrier DNA plus labeled probe atroom temperature. One of the three isolates, pNK162, hybridized to theoligo dT probe and was sequenced.

Using standard restriction digestion and subcloning techniques, NKSFclones pNK-6 and pNK162 were subcloned together in frame fortranscription and translation and ligated into the pXM expression vectorfor COS expression. The resultant clone, pNK40-4 (FIG. 1) is believed tocontain the full length cDNA for the 40 kD NKSF subunit.

EXAMPLE 6 EXPRESSION OF RECOMBINANT HUMAN NKSF

To produce NKSF, the DNAs encoding its subunits are transferred intoappropriate expression vectors, of which numerous types are known in theart for mammalian, insect, yeast, fungal and bacterial expression, bystandard molecular biology techniques. One such vector for mammaliancells is pXM [Y. C. Yang et al, Cel!, 47:3-10 (1986)]. This vectorcontains the SV40 origin of replication and enhancer, the adenovirusmajor late promoter, a cDNA copy of the adenovirus tripartite leadersequence, a small hybrid intervening sequence, an SV40 polyadenylationsignal and the adenovirus VA I gene, in appropriate relationships todirect the high level expression of the desired cDNA in mammalian cells[See, e.g., Kaufinan, Proc. Natl. Acad: Sci. USA, 82:689-693 (1985)].The pXM vector is linearized with the endonuclease enzyme XhoI andsubsequently ligated in equimolar amount separately to the cDNA encodingthe NKSF subunits that were previously modified by addition of syntheticoligonucleotides [Collaborative Research, Lexington, Mass.] thatgenerate Xho I complementary ends to generate constructs for expressionof each subunit of NKSF.

Another vector for mammalian expression, pEMC3 (1) can be made by simplemodification of the pEMC2B1 vector, described below. pEMC3 (1) differsfrom pEMC2B1 by three restriction sites, SmaI, SalI, XbaI, in thepolylinker region. To make pEMC3(1), these three restriction sites areinserted between the PstI and EcoRI restriction sites of pEMC2B I byconventional means.

pEMC2B1 may be derived from pMT2pc which has been deposited with theAmerican Type Culture Collection (ATCC), Rockville, Md. (USA) underAccession Number ATCC 40348. The DNA is linearized by digestion of theplasmid with PstI. The DNA is then blunted using T₄ DNA polymerase. Anoligonucleatide 5′ TGCAGGCGAGC CTGAATTCCTCGA 3′ (SEQ ID NO: 18) is thenligated into the DNA, recreating the PstI site at the 5′ end and addingan EcoRI site and XhoI site before the ATG of the DHFR cDNA. Thisplasmid is called pMT21. pMT21 is cut with EcoRI and XhoI which cleavesthe plasmid at two adjacent cloning sites. An EMCV fragment of 508 basepairs was cut from pMT₂ECAT₁ [S. K. Jong et al, J. Virol., 63:1651-1660(1989)] with the restriction enzymes EcoRI and TaqaI. A pair ofoligonucleotides 68 nuclectides in length were synthesized to duplicatethe EMCV sequence up to the ATG. The ATG was changed to an ATT, and a Cis added, creating a XhoI site at the 3′ end. A Taqal site is situatedat the 5′ end. The sequences of the oligonucleotides were: 5′CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGG ACGTGGTTTTCCTTTGAAAAACACGATTGC 3′(SEQ ID NO: 19) and its complementary strand.

Ligation of the pMT21 EcoRI-to-XhoI fragment to the EMCV EcoRI-to-TaqαIfragment and to the TaqaI/XhoI oligonucleotides produced the vectorpEMC2B 1. This vector contains the SV40 origin of replication andenhancer, the adenovirus major late promoter, a cDNA copy of themajority of the adenovirus tripartite leader sequence, a small hybridintervening sequence, an SV40 polyadenylation signal and the adenovirusVA I gene, DHFR and β-lactamase markers and an EMC sequence, inappropriate relationships to direct the high level expression of thedesired cDNA in mammalian cells.

The two different cDNAs are expressed simultaneously in the same host orindependently in different hosts. In the latter case, the subunits arepurified separately and the final active NKSF is assembled byrenaturation of the individual subunits.

a. Mammalian Cell Expression

To obtain expression of the NKSF protein for use in the assays describedbelow, the constructs containing the cDNAs for the 40 kD and 30 kD(smaller version) subunits were cloned separately into the mammalianexpression vector pEMC3(1) and together introduced into COS cells bycalcium phosphate coprecipitation and transfection. 35S methioninelabelled proteins (4 hr pulse, 2 days after transfection) ofapproximately 80 kD (nonreduced) and 40 kD and 30 kD (reduced) arepresent in PAGE gels of COS cotransfectant conditioned medium but not innegative control transfectants. The conditioned media from the COScotransfectants collected 48 hrs after transfection, was active in thegamma interferon (IFNγ) induction assay (see Example 7a).

Further evidence that the activity was identical to that purified from8866 conditioned medium comes from the observations that thecotransfected conditioned media synergizes with IL2 in the IFNγinduction assay and that polyclonal rabbit antiserum (1: 100 dilution)to the NKSF heavy chain, blocks the activity in the cotransfectant aswell as RPMI 8861 conditioned medium. The antiserum was produced byimmunizing rabbits with NKSF heavy chain purified from conditioned mediafrom COS cells transfected with the NKSF heavy chain cDNA (cloned inpEMC 3(1)).

When the pNK40-4 plasmid was separately transfected into COS cells, thesupernatant was collected and assayed, and the cells pulse labeled with³⁵S cysteine. The labeled protein was run on an 11% acrylamide gel understandard reducing and nonreducing conditions. The unlabeled supernatantfrom this transfection with pNK40-4 was inactive in the gamma interferoninduction assay and in the cell cytotoxicity assay, which were performedas described below in Example 8.

The mammalian cell expression vectors described herein maybe synthesizedby techniques well known to those skilled in this art. The components ofthe vectors, e.g. replicons, selection genes, enhancers, promoters, andthe like, may be obtained from natural sources or synthesized by knownprocedures. See, Kaufinan et al, J. Mol. Biol., 159:511-521 (1982); andKaufman, Proc. Natl. Acad. Sci.. USA 82:689-693 (1985). Exemplarymammalian host cells include particularly primate cell lines and rodentcell lines, including transformed cell lines. Normal diploid cells, cellstrains derived from In vitro culture of primary tissue, as well asprimary explants, are also suitable. Candidate cells need not begenotypically deficient in the selection gene so long as the selectiongene is dominantly acting. For stable integration of the vector DNAs,and for subsequent amplification of the integrated vector DNAs, both byconventional methods, CHO cells may be employed. Alternatively, thevector DNA may include all or part of the bovine papilloma virus genome[Lusky et al, Cell, 36:391-401 (1984)] and be carried in cell lines suchas C127 mouse cells as a stable episomal element. Other suitablemammalian cell lines include but are not limited to, HeLa, COS-1 monkeycells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIHmice, BHK or HaK hamster cell lines.

Where the two subunits require simultaneous expression in mammaliancells, the two cDNAs may be introduced into the cells using twodifferent selectable genes or markers. As discussed below in Example 7,this can readily be achieved in CHO cells using the dihydrofolatereductase (DHFR) gene as one marker and adenosine deaminase (ADA) as theother marker. Any combination of two genes which can be independentlyselected in any mammalian cell line are useful for this purpose. Forexample, a CHO cell line is independently developed for expression ofone subunit under ADA selection and a different cell line is developedfor expression of the other subunit under DHFR selection. The cell linesare fused in polyethylene glycol under double selection to yield stablelines expressing both subunits. Alternatively, the DNAs are introducedsimultaneously or sequentially into the same cells, thereby yieldinglines expressing active NKSF.

It is also possible that multicistronic vectors encoding both subunitswith a single selectable marker might yield cells in which both subunitscan be coamplified with one selective drug. Additionally, this effectmay be achieved by simple cotransfection of cells simultaneously withseparate vectors.

Stable transformants are then screened for expression of the product bystandard immunological, biological or enzymatic assays. The presence ofthe DNA and mRNA encoding the NKSF polypeptides may be detected bystandard procedures such as Southern blotting and RNA blotting.Transient expression of the DNA encoding the polypeptides during theseveral days after introduction of the expression vector DNA intosuitable host cells, such as COS-1 monkey cells, is measured withoutselection by activity or immunologic assay of the proteins in theculture medium.

One skilled in the art can also construct other mammalian expressionvectors comparable to the pEMC3 (1) vector by, e.g., inserting the DNAsequences of the NKSF subunits from the respective plasmids withappropriate enzymes and employing well-known recombinant geneticengineering techniques and other known vectors, such as pXM, pJL3 andpJL4 [Gough et al., EMBO J., 4:645-653 (1985)] and pMT2 (starting withpMT2-VWF, ATCC #67122; see PCT application PCT/US87/00033). Thetransformation into appropriate host cells of these vectors with bothNKSF subunits (either as separate vectors or in the same vector) canresult in expression of the NKSF polypeptides.

b. Bacterial Expression Systems

Similarly, one skilled in the art could manipulate the sequencesencoding the NKSF subunits by eliminating any mammalian regulatorysequences flanking the coding sequences and inserting bacterialregulatory sequences to create bacterial vectors for intracellular orextracellular expression of the NKSF subunits of the invention bybacterial cells. The DNA encoding the NKSF polypeptides may be furthermodified to contain different codons to optimize bacterial expression asis known in the art. Preferably the sequences encoding the mature NKSFsubunits are operatively linked in-frame to nucleotide sequencesencoding a secretory leader polypeptide permitting bacterial expression,secretion and processing of the mature NKSF polypeptides, also bymethods known in the art. The simultaneous expression of both subunitsof NKSF in E. coli using such secretion systems is expected to result inthe secretion of the active heterodimer. This approach has yieldedactive chimeric antibody fragments [See, e.g., Bitter et al, Science,240:1041-1043 (1988)].

Alternatively, the individual subunits are expressed in the mature formseparately from the two different cDNAs in E. coli using vectors forintracellular expression and the subunits are isolated separately, mixedand refolded by procedures well known in the art. See, for example, U.S.Pat. No. 4,512,922. The compounds expressed through either route inbacterial host cells may then be recovered, purified, and/orcharacterized with respect to physicochemical, biochemical and/orclinical parameters, all by known methods.

c. Insect or Yeast Cell Expression

Similar manipulations can be performed for the construction of an insectvector for expression of NKSF polypeptides in insect cells [See, e.g.,procedures described in published European patent application 155,476].If the NKSF subunits are derived from a single cDNA, this cDNA will beexpressed in insect cells. Alternatively, if the NKSF subunits arederived from two different cDNAs, each subunit is separately insertedinto an insect cell vector and the two resulting vectors co-introducedinto insect cells to express biologically active NKSF.

Similarly yeast vectors are constructed employing yeast regulatorysequences to express either the individual NKSF subunits simultaneously,or, if the protein is derived from a single precursor, the cDNA encodingthat precursor, in yeast cells to yield secreted extracellular activeNKSF heterodimer. Alternatively the individual subunits may be expressedintracellularly in yeast, the individual polypeptides isolated andfinally, refolded together to yield active NKSF. [See, e.g., proceduresdescribed in published PCT application WO 86/00639 and European patentapplication EP 123,289.]

EXAMPLE 7 CONSTRUCTION OF HO CELL LINES EXPRESSING HIGH LEVELS OF NKSF

One method for producing high levels of the NKSF protein of theinvention from mammalian cells involves the construction of cellscontaining multiple copies of the two cDNAs encoding the individual NKSFsubunits.

Because the two NKSF polypeptides are each derived from separate mRNAs,each corresponding cDNA must be expressed simultaneously in CHO cells.Two different selectable markers, e.g., DHFR and ADA, may be employed.One of the cDNAs is expressed using the DHFR system [Kaufman and Sharp,J. Mol. Biol., (1982) supra.] using, e.g., the vector pEMC3 (1) toexpress one of the NKSF subunits and DHFR. The second subunit isexpressed using a second vector, e.g. pMT3SV2ADA [R. J. Kaufinan, Meth.Enzymol., 185:537-566 (1990)]. Plasmid pMT3SV2ADA also directs theexpression of ADA in mammalian cells. The first vector constructcontaining one subunit is transfected into DHFR-deficient CHO DUKX-BIIcells. The second vector construct containing the second subunit istransfected into a second CHO cell line. The transfected cells areselected for growth in increasing concentrations of methotrexatebeginning with approximately 5 nM with subsequent step-wise incrementsup to 100 μM for the DHFR marker, or in 2′-deoxycoformycin (dCF) for theADA marker beginning with 100 nM with subsequent step-wise increments upto 10 μM. The expression of the individual cDNAs (one subunit under DHFRselection in one cell line and the other subunit under ADA selection ina second cell line) is assayed through a combination of mRNA blotting totest for transcription and immunoanalysis to test for proteinproduction. The cells which express one of the subunits under ADAselection and the cells which express the other subunit under DHFRselection are finally fused in polyethylene glycol using methods wellestablished in the art to yield a single cell line, resistant to bothdCF and MTX and expressing both subunits to yield biologically activeNKSF.

Another presently preferred method of expression is based on thedevelopment of a single cell line expressing both subunits. A firstvector containing one subunit, e.g., the first vector described above,is transfected into a selected CHO cell line and the expression of thesubunit is amplified under drug selection as described above. Thereafterthe second vector containing the other subunit is transfected into thecell line which already contains amplified first vector. The cDNAexpressing the other subunit, e.g., the second vector described above,may be introduced under a second drug selection. The second vector isthen amplified by the same techniques, resulting in a single cell lineexpressing both subunits simultaneously. (See, e.g., published PCTInternational Application WO88/08035 for an exemplary description ofindependently amplifying a first gene linked to a DHFR gene and a secondgene linked to an ADA gene.)

In another method, two vectors constructs may be designed, e.g., apEMC3(1) construct containing one subunit and the DHFR gene and a secondpEMC3 (1) construct containing the second subunit and the DHFR gene. Thetwo pEMC3 (1) constructs expressing both NKSF subunits may be mixed andthe mixture transfected into CHO cells. The cells are then amplified inMTX as described above to obtain a cell line producing both subunits.Alternatively two drug markers may be employed in this method and thecombined selection of both drugs may be used and transformants testedfor NKSF activity directly to obtain cell lines expressing theheterodimer.

Still a further alternative is the development of a multicistronicvector encoding both subunits and one drug selection marker.Transfection of this vector and its amplification might more rapidlyyield high expressing cell lines.

In any of the expression systems described above, the resulting celllines can be further amplified by appropriate drug selection, resultingcell lines recloned and the level of expression assessed using the gammainterferon induction assay described herein.

EXAMPLE 8 BIOLOGICAL ACTIVITIES OF HUMAN NKSF

The following assays were performed using either the homogeneous NKSFdescribed in Example 2 or a partially purified version of NKSF. Therecombinant version of the molecule is expected to exhibit NKSFbiological properties in these same assays or other assays.

When fresh human peripheral blood mononuclear cells (PBMC) orphytohemagglutinin (PHA)-induced blasts are cultured with NKSF,significant amounts of gamma interferon are detected in the supernatant.Moreover, NKSF synergizes with IL-2, phorbol dibutyrate (PdBu), and PHAin inducing gamma interferon production. Northern blot analyses showthat NKSF, alone or in combination with other factors, inducesaccumulation of gamma interferon mRNA. Gamma interferon message wasfound in both purified T and NK populations. Preincubation with theprotein synthesis inhibitor, cycloheximide (CHX), leads to asuperinduction of gamma interferon mRNA following stimulation with NKSF.HLA-DR(+) accessory cells are required for gamma interferon productionby T and NK cells. Induction of gamma interferon mRNA can be detected asearly as 1 hour after treatment with NKSF of PHA blasts. The details ofthe assay are described below.

a. Gamma Interferon Induction Assay

NKSF activity was measured by the induction of gamma interferon(gamma-FN) expression in cultures of human peripheral blood lymphocytes(PBLs). In the assay, 100 μl of human PBLs suspended (10⁷ cells/ml) inRPMI 1640 culture medium supplemented with 10% heat-inactivated FCS wasadded to 100 μl of sample to be tested in a microtiter plate [U-bottom,96-well, Costar, Cambridge, Mass.] and incubated for 18 hours at 37° C.,5% CO₂. Samples to be tested included purified NKSF, dialyzed cell freesupernatant from 48 hour phorbol diester stimulated RPMI 8866.cells, andrecombinant IL-2 [Genetics Institute, Inc., PCT application WO85/05124].After incubation, 100 μl of cell free supernatant was withdrawn fromeach well and the level of gamma-IFN produced measured byradioimmunoassay [Centocor Gamma Interferon Radioimmunoassay, Centocor,Malvern, Pa.]. One unit of NKSF per ml is the concentration required toproduce one-half of the maximal gamma-IFN produced in the presence ofoptimal concentrations of NKSF.

There was a linear positive correlation between the amount of gamma-IFNproduced in each well to the amount of NKSF in culture.

In addition to gamma-FN, NKSF induces T and NY cells to produce GM-CSFand tumor necrosis factor. The assay of production of these cytokines isperformed as above and the supernatant is assayed for the presence ofthe cytokines by specific biological assays or by radioimmunoassays[Cuturi et al, J. Exp. Med., 165:1581-1594 (1987)]. Alternatively, theinduction of the cytokine genes is measured by evaluating theaccumulation of mRNA transcripts of the three cytokines in thelymphocytes treated with NKSF. Lymphocytes are cultured for 4 to 18hours with NKSF, RNA is extracted by established methods, fractionatedby agarose gel electrophoresis, blotted on nitrocellulose, andhybridized with ³²P-labeled cDNA probes for the IFN-gamma, GM-CSF, ortumor necrosis factor genes (Northern blotting). Extent of hybridizationis determined by autoradiography and densitometry.

NKSF induces production of IFN-gamma and TNF from purified human NKcells. When assayed as described under the gamma interferon inductionassay of part (a) above, NK cells are able to lyse various target cellsby two mechanisms. One mechanism is spontaneous lysis, in the absence ofspecific sensitization, of a variety of target cells, includingleukemia- and solid tumor-derived cell lines, virus-infected cells, and,in some cases, normal cell lines. The second mechanism is ADCC.Preliminary evidence indicates that NKSF may enhance the ability of NKcells to lyse more efficiently target cells coated with IgG antibodieswith an Fc portion able to bind to the NK cell Fc receptor.

b. NK Assay

In order to assay for the enhancement of NK cell spontaneouscytotoxicity by NKSF, PBLs or purified NK cells (5×10⁶ cells/ml) areincubated for 18 hours in RPMI 1640 medium, 10% heat inactivated FCS, inthe presence of various dilutions of NKSF. PBLs are then washed andadded, at PBL-target cells ratio from 1:1 to 100:1, to 10⁴ ⁵¹Cr-labeledtarget cells in a U-bottomed microtiter plate (final volume 200 μl).After 4 hours, the plates are centrifuged, the cell-free supernatant iscollected and lysis of target cells is evaluated by the release of the⁵¹Cr-label from the cells. NKSF increases several-fold the cytotoxicityof NK cells when assayed against the following target cells: malignanthematopoietic cell lines (i.e. K562, Daudi, U937, HL-60, ML3, Molt 4,Jurkat, THP-1), solid tumor-derived cell line (rhabdomyosarcoma,melanoma), and normal foreskin-derived fibroblast strains. Theenhancement of NK cell-mediated cytotoxicity by NKSF is not secondary tothe production of IFN-gamma, tumor necrosis factor, or IL-2, produced bythe PBL treated with NKSF. The cytotoxic assay, the methods for NK cellpurification, and for the quantitative evaluation of enhancement of NKcell-mediated enhancement by cytokines are described in detail in G.Trinchieri et al, J. Exp. Med., 147:1314 (1978); G. Trinchieri et al, J.Exp. Med., 160:1147 (1984); and B. Perussia et al, Natural Immunity andCell Growth Regulation, 6:171-188 (1987).

c. ADCC Assay

In a standard antibody dependent cell mediated cytotoxity assay,preliminary results show that partially purified NKSF of the presentinvention enhanced NK cell killing of antibody coated tumor target cellsin a dose dependent manner. For antibodies capable of binding to the Fcreceptor of the NK cell, the ADCC response of NK cells was enhanced bythe addition of NKSF.

d. Co-Mitogenic Effect of NKSF

PBLs (0.5×10⁶/ml) are cultured in 200 μl of RPMI 1640 mediumsupplemented with 10% heat inactivated human AB serum. After 3 and 6days the PBLs are pulsed for 6 hours with ³H-thymidine and DNA synthesis(proliferation) is evaluated by the ³H-thymidine uptake in the cells bycollecting the cells on glass filters using a Skatron cell harvester andcounting the cell-associated H-Thymidine by liquid scintillation using aPackard Tricarb beta-counter. NKSF has minimal effect on PBLproliferation by itself, but is strongly co-mitogenic withphytohemagglutinin (PHA-M Welcome, 1:100) at day 6 of culture and withphorbol diesters (TPA or PDBu, 10⁻⁸ or 10⁻⁷M, respectively) at both day3 and day 6. Cell cycle analysis is performed by flow cytofluorometry(Cytofluorograf 50H, Ortho Diagnostics) using a technique combining DNAstaining with immunofluorescence staining according to London et al, J.Immunol., 137:3845 (1986). This analysis has shown that the PBLsaffected by the co-mitogenic effect of NKSF are T cells either CD4 orCD8 positive.

e. GM-CSF Induction Assay

Induction of GM-CSF expression in cultures of human PBLs was measured.In the assay, 100 μl of human PBLs suspended (10⁷ cells/ml) in RPMI 1640culture medium supplemented with 10% heat-inactivated FCS was added to100 μl of sample to be tested in a microtiter plate [U-bottom, 96-well,Costar, Cambridge, Mass.] and incubated for 18 hours at 37° C,. 5% CO₂.After incubation, 100 μl of cell-free supernatant was withdrawn fromeach well and the level of GM-CSF produced measured by enzyme-linkedimmunosorbent assay (ELISA) using two murine monoclonal antibodiesagainst human GM-CSF (3/8.20.5 and 2/3.1, supplied by GeneticsInstitutc, Inc.) recognizing different epitopes. Using recombinant humanGM-CSF (Genetics Institute, Inc.) as a standard, the detection limit ofthis assay was 50 μg/ml.

Numerous modifications and variations in practice of this invention areexpected to occur to those skilled in the art.

1-31. (canceled)
 32. Isolated DNA sequences encoding natural killer cellstimulatory factor protein exhibiting natural killer cell stimulatoryprotein activity, wherein said DNA sequences comprise: a) a mutatednucleic acid encoding a protein comprising amino acids 23 to 328 of theamino acid sequence of FIG. 1, wherein said amino acids have asubstitution and/or deletion mutation in a glycosylation site chosenfrom asn-X-thr or asn-X-ser, wherein X is any amino acid, and b) nucleicacid encoding a protein comprising amino acids 57 to 253 of the aminoacid sequence of FIG.
 2. 33. The mutated nucleic acid of claim 31,wherein the mutation in the glycosylation site results in partialglycosylation at that site.
 34. The mutated nucleic acid of claim 32,wherein the mutation in the glycosylation site results in noglycosylation at that site.
 35. The mutated nucleic acid of claim 32,wherein the X in the glycosylation site is deleted.
 36. The mutatednucleic acid of claim 32, wherein the asn in the glycosylation site isdeleted or substituted with another amino acid.
 37. The mutated nucleicacid of claim 32, wherein the thr or ser in the glycosylation site isdeleted or substituted with another amino acid, wherein when theglycosylation site contains a thr it is not mutated to a ser and whenthe glycosylation site contains a ser it is not mutated to a thr. 38.Isolated DNA sequences encoding natural killer cell stimulatory factorprotein exhibiting natural killer cell stimulatory protein activity,wherein said DNA sequences comprise: a) a nucleic acid encoding aprotein comprising the amino acids 23 to 328 of the amino acid sequenceof FIG. 1, and b) a mutated nucleic acid encoding a protein comprisingamino acids 57 to 253 of the amino acid sequence of FIG. 2, wherein saidamino acids have a substitution and/or deletion mutation in aglycosylation site chosen from asn-X-thr or asn-X-ser, wherein X is anyamino acid.
 39. The mutated nucleic acid of claim 38, wherein themutation in the glycosylation site results in partial glycosylation atthat site.
 40. The mutated nucleic acid of claim 38, wherein themutation in the glycosylation site results in no glycosylation at thatsite.
 41. The mutated nucleic acid of claim 38, wherein the X in theglycosylation site is deleted.
 42. The mutated nucleic acid of claim 38,wherein the asn in the glycosylation site is deleted or substituted withanother amino acid.
 43. The mutated nucleic acid of claim 38, whereinthe thr or ser in the glycosylation site is deleted or substituted withanother amino acid, wherein when the glycosylation site contains a thrit is not mutated to a ser and when the glycosylation site contains aser it is not mutated to a thr.
 44. Isolated DNA sequences encodingnatural killer cell stimulatory factor protein exhibiting natural killercell stimulatory protein activity, wherein said nucleic acids comprise:a) a mutated nucleic acid sequence comprising nucleotides 99 to 1016 ofthe nucleotide sequence of FIG. 1 wherein said nucleic acid sequence hasa substitution and/or deletion mutation in a sequence encoding aglycosylation site chosen from asn-X-thr or asn-X-ser, wherein X is anyamino acid, and b) a mutated nucleic acid sequence comprisingnucleotides 269 to 859 of the nucleotide sequence of FIG. 2, whereinsaid nucleic acid sequence has a substitution and/or deletion mutationin a sequence encoding a glycosylation site chosen from asn-X-thr orasn-X-ser, wherein X is any amino acid.
 45. The nucleic acid sequencesof claim 32, wherein said nucleic acid sequences are in operativeassociation of an expression control sequence.
 46. The nucleic acidsequences of claim 39, wherein said nucleic acid sequences are inoperative association of an expression control sequence.
 47. The nucleicacid sequences of claim 44, wherein said nucleic acid sequences are inoperative association of an expression control sequence.
 48. A cellcomprising the nucleic acid sequences of claim
 45. 49. A cell comprisingthe nucleic acid sequences of claim
 46. 50. A cell comprising thenucleic acid sequences of claim
 47. 51. A process for producing naturalkiller cell stimulatory protein, comprising the steps of: a) culturingthe host cell of claim 48; and b) isolating said protein.
 52. A processfor producing natural killer cell stimulatory protein, comprising thesteps of: a) culturing the host cell of claim 49; and b) isolating saidprotein.
 53. A process for producing natural killer cell stimulatoryprotein, comprising the steps of: a) culturing the host cell of claim50; and b) isolating said protein.
 54. An isolated DNA sequence thathybridizes to a nucleic acid of claim 32 under stringent conditionschosen from: a) 4×SSC at 65° C., followed by washing in 0.1×SSC at 65°C. for one hour; and b) 50% formamide in 4×SSC at 42° C.
 55. An isolatedDNA sequence that hybridizes to a nucleic acid of claim 44 understringent conditions chosen from: a) 4×SSC at 65° C., followed bywashing in 0.1×SSC at 65° C. for one hour; and b) 50% formamide in 4×SSCat 42° C.